Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.


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Page 2 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8SDW 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: X-RAY DIFFRACTION The reported resolution of this entry is 1.75 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic.The table shows the number of entries on which the scores are based.The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density.The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively.A grey segment represents the fraction of residues that are not modelled.

Metric
The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density.The numeric value is given above the bar.

Entry composition i ○
There are 4 unique types of molecules in this entry.The entry contains 1651 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.

Mol Chain Residues Atoms ZeroOcc AltConf
Trace There is a discrepancy between the modelled and reference sequences: • Molecule 2 is GUANOSINE-3'-MONOPHOSPHATE-5'-DIPHOSPHATE (three-letter code: G3D) (formula: C 10 H 16 N 5 O 14 P 3 ) (labeled as "Ligand of Interest" by depositor).3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry.The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and electron density.Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.
• Molecule 1: ADP-ribosylation factor 1 Chain A: There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms).The all-atom clashscore for this structure is 3.
All (8) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude.

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Page 8 Full There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.no such sidechains identified.

RNA i ○
There are no RNA molecules in this entry.
5.4 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligand geometry i ○
Of 2 ligands modelled in this entry, 1 is monoatomic -leaving 1 for Mogul analysis.
In the following There are no bond angle outliers.
There are no chirality outliers.
All (4) torsion outliers are listed below: There are no ring outliers.
No monomer is involved in short contacts.
The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances the Ligand of Interest.In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included.For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identified as an outlier by Mogul will be highlighted in the graph.For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identified by Mogul is calculated over all ring torsion angles.If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identified rings is also greater than 60 degrees, then that ring is considered an outlier.The outliers are highlighted in purple.The color gray indicates Mogul did not find sufficient equivalents in the CSD to analyse the geometry.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution.The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue.The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligands i ○
In the following The following is a graphical depiction of the model fit to experimental electron density of all instances of the Ligand of Interest.In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included.Each fit is shown from different orientation to approximate a three-dimensional view.
Electron density around G3D A 201: and green (positive)

Other polymers i ○
There are no such residues in this entry.
are no protein residues with a non-rotameric sidechain to report.Sometimes sidechains can be flipped to improve hydrogen bonding and reduce clashes.There are 2mF o -DF c (at 0.7 rmsd) in gray mF o -DF c (at 3 rmsd) in purple (negative) and green (positive) MG A 202: 2mF o -DF c (at 0.7 rmsd) in gray mF o -DF c (at 3 rmsd) in purple (negative)

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• Molecule 3 is MAGNESIUM ION (three-letter code: MG) (formula: Mg) (labeled as "Ligand of Interest" by depositor).F

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e wBond lengths and bond angles in the following residue types are not validated in this section: G3D, MGThe Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).
In the following table, the Chirals column lists the number of chiral outliers, the number of chiral centers analysed, the number of these observed in the model and the number defined in the Chemical Component Dictionary.Similar counts are reported in the Torsion and Rings columns.'-' means no outliers of that kind were identified.Continued on next page...
table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary.The Link column lists molecule types, if any, to which the group is linked.The Z score for a bond length (or angle) is the number standard deviations the observed value is removed from the expected value.A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).All (3) bond length outliers are listed below: Page 13Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8SDW median, 95 th percentile and maximum values of B factors of atoms in the group.The column labelled 'Q< 0.9' lists the number of atoms with occupancy less than 0.9.Mol Type Chain Res Atoms RSCC RSR B-factors(Å 2 ) Q<0.9 table, the Atoms column lists the number of modelled atoms in the group and the number defined in the chemical component dictionary.The B-factors column lists the minimum,F o r M a n u s c r i p t R e v i e w